Non-segmented negative-sense (NNS) RNA viruses include many of our most pathogenic and sometimes deadly viruses, such as measles, rabies, Ebola, and respiratory syncytial virus (RSV). Unfortunately, no effective vaccine or antiviral therapy is available to prevent or treat infection by RSV and many other NNS RNA viruses. Therefore, there is a critical need to define the structural and molecular basis of RNA synthesis of NNS RNA viruses and any differences between them. Our long-term goal is to understand the RNA synthesis machinery of NNS RNA viruses and facilitate the development of antiviral drugs. Our overall objectives in this application are to elucidate the molecular mechanisms of the RSV polymerase and provide functional and structural insights into RSV RNA synthesis. Our underlying hypothesis is that the catalytic activities of RNA polymerization, cap addition, and cap methylation reside within the RSV L protein, and L requires a dynamic assembly with its cofactors P and M2-1 to coordinate these activities during RNA synthesis. The rationale for this project is that understanding the mechanism of the RSV RNA synthesis is likely to offer strong scientific frameworks whereby new strategies to investigate related NNS RNA viruses can be developed. To test the central hypothesis, we will define the functional organization of the RSV L protein and determine the structure of the RSV polymerase using cryo-EM. We will also define the regulatory mechanisms of the RSV transcription by M2-1. These results are expected to have a broader impact beyond RSV because the shared strategies of RNA synthesis machines among NNS RNA viruses imply that our findings will be relevant to all members of this order. This proposed research program is innovative, in the applicant?s opinion, because the proposed research will lay solid foundations for in-depth mechanistic studies of the novel enzymatic activities of NNS RNA viruses and define novel structural and biochemical features of the RSV L protein as well as provide novel insights on the regulation of RSV RNA synthesis. The proposed research is significant because it leverages the power of interdisciplinary approaches that include single particle cryo-EM to open new horizons for visualizing key stages of RSV RNA synthesis effectively. Ultimately, such knowledge has the potential of offering new opportunities for the rational design of novel antiviral drugs to treat the devastating diseases that RSV and related NNS RNA viruses cause.